In order to analyze inorganic or organic molecules, especially biomolecules such as peptides, proteins, DNA, natural products, e.g., useful as drugs, metabolic intermediates, lipids and the like, it is usually required to isolate a compound of interest from a mixture prior to a downstream analysis so as to gain useful analytical data.
The isolation and subsequent analysis/characterization of, e.g., proteins, has been attempted by combining high-resolution separation techniques applied to complex protein mixtures with state-of-the-art identification methods such as mass spectrometry (MS). It is generally agreed that none of the existing separation and identification methodologies on its own can give a full account of the protein composition or the protein expression in complex mixtures, (e.g. biological matrices, biological fluids such as serum, plasma, synovial fluid, cerebrospinal fluid, urine, whole cells, cell fractions, cell lysates, or tissue extracts). This limitation, however, has not prevented the use of existing methods (or the combination of several existing technologies) to provide valuable information on a wide range of proteins, especially when either their absence or presence, or their level of expression can be correlated to a disease state.
One of the major barriers to widely applicable, e.g., MS analyses, NMR analyses, circular dichroism analyses, determination of X-ray diffraction patterns after crystallization or UV-spectroscopy analyses of biological samples is the successful purification or at least substantial enrichment of the molecules of interest to make them suitable for the subsequent analysis. A hurdle to overcome in the discovery phase of proteins or other compounds is the fact that the analytical tools used at the end of the process chain such as MS have a definite detection limit for finite amounts of proteins (or peptides derived thereof). To fully exploit the sensitivity limits and purification requirements for, e.g., peptide identification by MS, it is necessary to separate and/or enrich the protein of interest from a mixture of proteins or other bioorganic mixture.
Electrophoresis is a well-established technology for separating particles based on the migration of charged particles under the influence of a direct electric current. Several different operation modes such as isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP) have been developed as variants of the above separation principle and are generally known to those of skill in the art.
Free-flow electrophoresis (FFE) is a technology wherein the separation of the analytes occurs in liquid medium in the absence of a stationary phase (or solid support material). FFE is often referred to as carrier-less deflection electrophoresis or matrix-free deflection electrophoresis.
In the field of proteomics, FFE is the technology of choice for the defined separation of complex protein samples in terms of their varying isoelectric point (pI) values. Using FFE, organic and inorganic molecules, bioparticles, biopolymers and biomolecules can be separated on the basis of their electrophoretic mobility. The corresponding principles have already been described [e.g. Bondy B. et al. (1995), “Sodium chloride in separation medium enhances cell compatibility of free-flow electrophoresis”, Electrophoresis 16: 92-97].
The process of FFE has been improved in recent years, e.g., by way of stabilization media and counter-flow media. This is reflected, for example, in U.S. Pat. No. 5,275,706, the disclosure of which is hereby incorporated by reference in its entirety. According to this patent, a counter-flow medium is introduced into the separation space counter to the continuous flow direction of the bulk separation medium and sample that travels between the electrodes. Both media (separation media and counter flow media) are discharged or eluted through fractionation outlets into suitable collection devices such as a micro titer plate, resulting in a fractionation process having a low void volume. Additionally, a laminar flow of the media in the region of the fractionation outlets is maintained (i.e., with very low or no turbulence).
A particular FFE technique referred to as interval FFE is disclosed, for example, in U.S. Pat. No. 6,328,868. In this patent, the sample and separation medium are both introduced into an electrophoresis chamber, and the analytes in the sample are separated using an electrophoresis mode such as zone electrophoresis (ZE), isoelectric focusing (IEF) or isotachophoresis (ITP), and are finally expelled from the chamber through fractionation outlets. Embodiments of the '868 patent describe the separation media and sample movement to be unidirectional, traveling from the inlet end towards the outlet end of the chamber, with an effective voltage applied causing electrophoretic migration to occur while the sample and media are not being fluidically driven from the inlet end towards the outlet end, in contrast to the technique commonly used in the art wherein the sample and media pass through the apparatus while being separated in an electrical field (commonly referred to as continuous FFE).
The so-called cyclic mode or cyclic interval mode in the context of FFE as used herein has been described in International application PCT/EP2007/059010 (claiming priority from U.S. provisional applications U.S. Ser. No. 60/823,833 and U.S. Ser. No. 60/883,260), which is hereby incorporated by reference in its entirety. In sum, the cyclic interval mode is characterized by at least one, and possible multiple reversals of the bulk flow direction while the sample is being kept in the electrophoretic field between the elongated electrodes. In contrast to the static interval mode, the sample is constantly in motion thereby allowing higher field strength and thus better (or faster) separation. Additionally, by reversing the bulk flow of the sample between the elongated electrodes, the residence time of the analytes in the electrical field can be increased considerably, thereby offering increased separation time and/or higher separation efficiency and better resolution. The reversal of the bulk flow into either direction parallel to the elongated electrodes (termed a cycle) can be repeated for as often as needed in the specific situation, although practical reasons and the desire to obtain a separation in a short time will typically limit the number of cycles carried out in this mode.
A number of separation media for the separation of analytes such as bioparticles and biopolymers are known in the art. For example, the book “Free-flow Electrophoresis”, published by K. Hannig and K. H. Heidrich, (ISBN 3-921956-88-9) reports a list of separation media suitable for FFE and in particular for free-flow ZE (FF-ZE).
U.S. Pat. No. 5,447,612 discloses another separation medium which is a pH buffering system for separating analytes by isoelectric focusing by forming functionally stable pre-cast narrow pH zone gradients in free solution. It employs buffering components in complementary buffer pairs.
U.S. Pending Provisional Ser. No. 60/945,246 refers to volatile buffer systems suitable for FFE. The volatile buffer systems offer the advantage that they can be easily removed subsequent to a FFE step and prior to a downstream analysis such as MS, or do not disturb a downstream analysis.
Unfortunately, a variety of proteins, which have recently come into focus of today's research, are insoluble or nearly insoluble in aqueous solutions. In those methods, wherein surfactants, e.g., sodium dodecylsulfate (SDS), urea or Triton X-100 are commonly used to prevent proteins from precipitation, e.g., during electrophoresis the surfactants must be removed prior to a subsequent analysis such as MS because these surfactants interfere with the sensitivity of mass spectrometry detection. In order to address this problem, classical methods to remove such disturbing substances are normally used subsequent to a successful electrophoresis and prior to, e.g., a downstream MS analysis. These extensive and often difficult cleaning and/or purification procedures cause an increase in the overall length of time for the analysis and typically result in a loss of sample. Furthermore, these laborious procedures represent an obstacle for automation. Therefore, sample preparation is one critical, and often technically challenging task in a successful biomolecule analysis project today. There is a need for surfactants which allow a separation of (an) analyte(s) of interest from a mixture of analytes without the need of extensive time and sample consuming purification steps to remove said surfactants after the separation step. There are surfactants known in the art which are essentially suitable for mass spectrometric analysis. Non-ionic surfactants such as octyl-β-glucopyranoside have been used for mass spectrometric applications (e.g., Hatt, P et al., 1997). However, the electrophoretic separation still requires SDS and a time consuming surfactant exchange step is needed.
Norris et al. (Anal Chem. 75(23), 6642-7, 2003) describe the properties of an acid labile “cleavable detergent” named 3-[3-(bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) and the use of the compound during extraction of proteins and in MS analysis, but the analysis was carried out with the crude extract without a separation method to provide a separated or at least partially substantially analyte of interest.
In 2005, Norris et al. (J. Mass Spectrom., 1319-1326, 2005) describes further “cleavable detergents” which increase the solubility of proteins and which are photo labile or fluoride cleavable instead of acid labile. After cleavage of the detergents the moieties may precipitate and they no longer disturb MS analysis, or can be easily removed prior to MS analysis.
US patent application US 2006/0292607, related to Norris et al., describes methods for analyzing tissue from the surgical margin of resected tumor and describes the use of the cleavable detergent 3-[3-(bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to extract proteins contained within the interior of a cell and a subsequent MS analysis thereof.
A second publication of Norris et al. in 2005 (Anal. Chem., 77, 5036-5040, 2005) is directed to the use of cleavable detergents in mass spectrometry, wherein said detergents comprise a moiety which may act as an MS-matrix after cleavage of the detergent.
In US 2006/0240562 and WO 02/097393, cleavable compositions and methods employing such compositions, especially in MALDI-MS analysis of hydrophobic proteins, are described.
WO 2006/047614 describes MS-compatible solubilizers, which can increase the solubility of an analyte and the use thereof. The solubilizers are described to be suitable for LC/MS.
WO 00/70334 and WO 03/102536 describe destructible surfactants and methods of using them. The surfactants contain dioxolane or dioxane functional groups which enables the surfactant to be broken down under acidic conditions. The surfactants described herein are anionic, which makes them unattractive for FFE methods, especially for free-flow isoelectric focusing (IEF) in view of the charge added to the particles in the sample to be separated.
Ying-Qing Yu et al. (Anal. Chem., 75, 6023-6028, 2003) describe acid labile anionic surfactants for in-solution enzymatic digestion of proteins. The surfactants are suitable for HPLC-MS. Because of the anionic character of the surfactants, they are not suitable for FFE methods.